This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are cAMP (cyclic adenosine 5xe2x80x2-monophosphate) phosphodiesterases, and fragments thereof derived from mammals, especially humans, rats, and mice, and most especially from the brains, for example, the hippocampal and other memory related regions. Both human and rat isoforms are specifically disclosed herein. The methods according to the present invention disclose uses of this polypeptide in diagnostic assays and for screening potential therapeutic agents, as well as other potential uses.
The phosphodiesterases (PDEs) represent a family of enzymes that catalyze the hydrolysis of the various cyclic nucleoside monophosphates (including cAMP). These cyclic nucleotides have been found to act as second messengers within the cells, which messengers carry impulses from cell surface receptors having bound various hormones and neurotransmitters. The job of phosphodiesterases is to degrade these cyclic mononucleotides once their messenger role is completed (thereby regulating the level of cyclic nucleotides within the cells and maintaining cyclic nucleotide homeostasis).
A number of different families of such phosphodiesterases have been identified, with the predominant family having been designated PDE4, as distinguished by various kinetic properties (such as low Michaelis constant for cAMP and sensitivity to certain drugs). [See: Wang et al., Expression, Purification, and Characterization of human cAMP-Specific Phosphodiesterase (PDE4) Subtypes A, B, C, and D, Biochem. Biophys. Res. Comm., 234, 320-324 (1997)] Since the PDEs have been found to possess distinct biochemical properties it is likely that they are subject to a variety of different forms of regulation.
The phosphodiesterases disclosed according to the present invention exist in mammals in the form of isoenzymes (which represent different molecular forms of the same enzyme polypeptide). These phosphodiesterases (denoted PDE4D) are localized in the cytosol of the cell and are unassociated with any known membranous structures. The PDE4D isoenzymes specifically degrade cAMP and are a common target for such pharmacological agents as antidepressants (for example, rolipram). Also, inhibitors of PDE4 isoenzymes are powerful anti-inflammatory agents and may also be useful as anti-asthmatics.
In the past, attempts at isolation of such proteins have proven difficult since they are commonly present at very low concentration and have shown a tendency toward instability on purification. [See: Salanova et al., Heterologous Expression and Purification of Recombinant Rolipram-Sensitive Cyclic AMP-Specific Phosphodiesterases, Methods: A Companion to Methods in Enzymology 14, 55-64 (1998)]
Cloning of the isoenzymes of PDE4 have shown that mammals have up to 4 genes for PDE4, and this is true of rats, mice and humans. Further, each such gene has been found to code for several different protein variants. Thus, each PDE4 gene has been found to code for at least 2 or more polypeptides. The physiological roles for this plethora of forms of PDE4 is beginning to be understood with the advent of Recombinant DNA technology and the use of cDNAs to clone the various polypeptide forms of these enzymes. Such methodology is essential to the successful screening of drugs having, for example, anti-inflammatory activity.
The present invention is directed to novel polypeptides, with or without associated phosphodiesterase activity, and containing a novel amino acid sequence as part of the overall sequence. The present invention is also directed to novel cDNA clones coding for such polypeptides and sequences of DNA contained within the genome and which may code for such novel polypeptide sequences.
The present invention is also directed to novel cells and cell lines containing polynucleotides that make them capable of expressing the novel polypeptides disclosed herein.
It is one object of the present invention to provide methods of forming such cell lines by incorporating novel DNA sequences of the invention in vectors that can be used to transform cells into cells that express the polypeptides containing the novel sequences disclosed herein. Such polypeptides will often have phosphodiesterase activity that will affect the level of cAMP within the transformed cells so that the cAMP level will provide an indicator of the level of phosphodiesterase within such cells.
It is a further object of the present invention to provide such vectors, containing the novel DNAs disclosed herein, and genetically transformed cells capable of expressing the polypeptides coded for by said novel DNAs.
In the brain, the level of cAMP within neurons is believed to be related to the quality of memory, especially long term memory. Thus, since PDE4D degrades cAMP, the level of this enzyme could have effects on memory in animals, for example, in humans.
It is therefore another object of the present invention to monitor the levels of the novel polypeptides disclosed herein as a means of determining the presence of a disease condition or susceptibility to such condition, especially where such condition involves loss of memory, most especially long term memory.
It is a still further object of the present invention to provide novel DNA sequences present in cells, especially brain cells, that can serve as the target for probes capable of disclosing the presence of a mutation in said novel DNA sequences, whereby the presence of such a mutation indicates a possible cause for over- or under-activity of a novel phosphodiesterase as disclosed herein.
It is also a further object of the present invention to provide a method of using the transformed cells disclosed herein as a means of screening potential chemical agents, either small molecules or otherwise, for ability to inhibit the actions of phosphodiesterase polypeptides disclosed herein and thereby provide novel therapeutic agents for the treatment of diseases, for example, impairment of memory, especially long term memory, or as prophylactic agents to be used in anticipation of such conditions, perhaps as secondary conditions incident to already existing diseases.
Finally, it is an additional object of the present invention to provide transgenic animals, especially transgenic mice, into whose genome has been inserted a gene, present as one or more copies thereof, coding for the human isoform of the PDE4D6 of the invention, especially where this gene replaces the mouse gene otherwise coding for any corresponding isoform, and most especially where the effect of such humanization is the overproduction of the human PDE4D6 isoform in said mouse. The invention also relates to the production of xe2x80x9cknock-outxe2x80x9d animals, especially xe2x80x9cknock-outxe2x80x9d mice, whose genes for PDE4D6 isoforms have been rendered non-functional so that said mice produce no. PDE4D6 phosphodiesterase. Such mice thereby provide means of studying the effects of over-production, or no production, of the PDE4D6 isoforms disclosed according to the invention.